PO-040 Characterisation of cdk12 knocked out ovarian cancer cell lines

Introduction While cyclin-dependent kinases (CDKs) have a key role in promoting/controlling transition between the different phases of the cell cycle, transcriptional kinases, like CDK12, are mainly involved in gene transcription. CDK12 has been shown to regulate the expression of genes involved in DNA damage and to maintain genomic stability. Impairment of CDK12 activity is synergic with PARP inhibitor and cisplatin treatments in different cellular systems. We here aimed to generate ovarian cancer cell lines knocked out (KO) for CDK12 to understand its role in ovarian cancer and in response to chemotherapy. Material and methods A2780 and SKOV3 CDK12 KO clones were generated with CRISPR/Cas9 technology. Cell cycle analysis was evaluated by standard flow cytometric methods and DNA repair genes levels by Real Time PCR. Caspase 3 activity was measured to detect apoptosis with a luminescence-based assay. Cytotoxicity experiments were performed treating cells with different drug concentrations and evaluating cell survival after 72 hours by MTS assay. For in vivo studies 7.5 millions of cells were transplanted subcutaneously in nude mice and animals were monitored for tumour appearance and growth. Results and discussions We obtained 2 CDK12 KO ovarian cancer clones, A2780 KO and SKOV3 KO, out of more than 300 clones screened. The cell growth of both A2780 KO and SKOV3 KO cells is slower than the wild type (WT) cells, they have a less clonogenic ability and a tetraploid DNA content. Both CDK12 KO clones have a higher basal caspase activity than the WT cell lines, indicative of higher basal induction of apoptosis, while no increase in autophagy or senescence is observed. Both CDK12 KO clones show a decreased expression in BRCA1 and FANCD2 DNA repair genes than the WT cells. Cytotoxic experiments with anticancer agents with different mechanism of action show that both KO clones are less sensitive to ATM, CHK1 and WEE1 inhibitors treatment as compared to WT cells, while platinum and PARP inhibitors show similar cytotoxic activity in KO and WT cells. Interestingly enough, when KO clones were transplanted in nude mice, no tumour take was observed. Conclusion We were able to obtain CDK12 KO cells. We think that these models could help in disclosing new roles of CDK12 in ovarian carcinoma and may represent a useful tool to study new combination therapies for tumours with CDK12 mutations

Characterization of MTAP gene expression in breast cancer patients and cell lines

MTAP is a ubiquitously expressed gene important for adenine and methionine salvage. The gene is located at 9p21, a chromosome region often deleted in breast carcinomas, similar to CDKN2A, a recognized tumor suppressor gene. Several research groups have shown that MTAP acts as a tumor suppressor, and some therapeutic approaches were proposed based on a tumors\ub4 MTAP status. We analyzed MTAP and CDKN2A gene (RT-qPCR) and protein (western-blotting) expression in seven breast cancer cell lines and evaluated their promoter methylation patterns to better characterize the contribution of these genes to breast cancer. Cytotoxicity assays with inhibitors of de novo adenine synthesis (5-FU, AZA and MTX) after MTAP gene knockdown showed an increased sensitivity, mainly to 5-FU. MTAP expression was also evaluated in two groups of samples from breast cancer patients, fresh tumors and paired normal breast tissue, and from formalin-fixed paraffin embedded (FFPE) core breast cancer samples diagnosed as Luminal-A tumors and triple negative breast tumors (TNBC). The difference of MTAP expression between fresh tumors and normal tissues was not statistically significant. However, MTAP expression was significantly higher in Luminal-A breast tumors than in TNBC, suggesting the lack of expression in more aggressive breast tumors and the possibility of using the new approaches based on MTAP status in TNB

Multi-Chemotherapeutic Schedules Containing the pan-FGFR Inhibitor ARQ 087 are Safe and Show Antitumor Activity in Different Xenograft Models

ARQ 087 is a multi-tyrosine kinase inhibitor with potent activity against the FGFR receptor family, currently in Phase I clinical studies for the treatment of advanced solid tumors. The compound has a very safe profile and induces tumor regressions in FGFR-driven models. The feasibility of combining ARQ 087 with chemotherapy was investigated in FGFR deregulated human xenografts. Nude mice were transplanted subcutaneously with H1581, and when tumor masses reached 150 mg, were randomized to receive vehicle, ARQ 087, paclitaxel, carboplatin as single agents or in combination. Similar experimental conditions were applied in nude mice bearing SNU16 and MFE296 xenografts, with the inclusion of capecitabine in the former xenograft model. In the different xenograft models, the drugs given as single agents ranged from very active to partially active. The double combinations were more active than the single ones, but the triple combinations were the most active. In particular, the combination of ARQ 087 + paclitaxel + carboplatin in H1581 bearing mice was able to induce tumor regression in all the mice, with 6/8 mice tumor free at day 140 after tumor transplant. Of note, no toxic deaths nor premature stopping or delaying of drug administration were observed. The data herein reported demonstrated the feasibility of using xenografts models for poli-chemotherapeutic trials mimicking the best standard of care in treatment of specific tumor type and that ARQ 087, a new pan-FGFR inhibitor, can be safely combined with standard cytotoxic chemotherapeutic drugs with apparently no sign of cumulative toxicity and an associated increased antitumor effect

Combination of the c-Met Inhibitor Tivantinib and Zoledronic Acid Prevents Tumor Bone Engraftment and Inhibits Progression of Established Bone Metastases in a Breast Xenograft Model

<div><p>Bone is the most common metastatic site for breast cancer. There is a significant need to understand the molecular mechanisms controlling the engraftment and growth of tumor cells in bone and to discover novel effective therapeutic strategies. The aim of this study was to assess the effects of tivantinib and Zoledronic Acid (ZA) in combination in a breast xenograft model of bone metastases. Cancer cells were intracardially implanted into immunodeficient mice and the effects of drugs alone or in combination on bone metastasis were evaluated by <i>in vivo</i> non-invasive optical and micro-CT imaging technologies. Drugs were administered either before (preventive regimen) or after (therapeutic regimen) bone metastases were detectable. In the preventive regimen, the combination of tivantinib plus ZA was much more effective than single agents in delaying bone metastatic tumor growth. When administered in the therapeutic schedule, the combination delayed metastatic progression and was effective in improving survival. These effects were not ascribed to a direct cytotoxic effect of the combined therapy on breast cancer cells <i>in vitro</i>. The results of this study provide the rationale for the design of new combinatorial strategies with tivantinib and ZA for the treatment of breast cancer bone metastases.</p></div

Effect of combination on <i>in vitro</i> cytotoxicity and cell migration.

<p>(<b>A</b>) In vitro cytotoxic activity of tivantinib alone (▪) or in combination with ZA (□) against 1833/TGL cells. Values represent the percentage of controls. (<b>B</b>) 1833/TGL cells were seeded at the concentration of 1×10⁵ cells/mL on a 12-well culture plate in six-well plates. On day 0, for each well, a wound was made in the center of the monolayer of confluent cells with a sterile plastic pipette tip and vehicle or drugs were added. The plate was placed under a motorized inverted microscope and wounds were photographed at 0 (<i>t</i>₀), 24, and 48 hours after wounding. Representative photographs of a filed of view from the different experimental conditions are shown. (<b>C</b>) Quantitative analysis: the level of cell migration was quantified as the percentage of wound closure at each time point after the wound scratch. Values represent averages ± SE of two independent experiments, each consisting of 2 replicates. Statistical analysis were performed at 24 and 48 hours after wounding.</p

Scheme showing the experimental protocol used.

<p>Scheme showing the experimental protocol used.</p

<i>In vivo</i> effects of tivantinib and ZA alone and in combination in a therapeutic schedule of bone metastases.

<p>The bone metastatic 1833/TGL cells were injected into the left cardiac ventricle of 4-week old athymic nude mice (5×10⁵ cells/mouse). When bone metastases were detectable (11 days after cell implant), implanted animals were randomized into 4 groups of 10 mice each: controls treated with vehicle PEG 400∶20% vitamin E TPGS solution (60∶40) or treated groups administered with tivantinib alone (300 mg/Kg), ZA (100 mg/Kg) alone and combination of tivantinib plus ZA. Both vehicle and tivantinib were administered daily <i>per os</i> starting 13 days after implant until the end of the experiment. ZA was administered i.p. every 2 days starting 13 days after implant till the end of the experiment. (<b>A</b>) Representative optical scanning in the supine position for each group 27 days after tumor implant. The intensity of the BLI signal, measured as total photon flux, is shown as a pseudo-color scale bar. Numbers at the left bottom of the images represent the number of mice. (<b>B</b>) At the indicated times after xenografting, the BLI signal was captured and the growth kinetics of right and left hindlimb metastasis for each group were expressed in the graph as mean value of total photon counts from the hindlimbs of animals. Data are presented as mean value ± SE; <b>*</b>, p<0.05 tivantinib+ZA vs tivantinib. (<b>C</b>) Representative volume-rendered micro-CT images of osteolytic bone metastases obtained 27 days after xenografting are shown. Red circles indicate osteolysis and the numbers at the bottom of the images represent the number of mice. (D) Kaplan–Meyer curves of vehicle and treated groups.</p

<i>In vivo</i> effects of tivantinib and ZA single agent and in combination in a preventive schedule of bone metastases.

<p>(<b>A–B</b>) <i>In vivo</i> bioluminescence imaging (BLI) of 1833/TGL-i.c. injected athymic nude mice (4 week old) treated with tivantinib (120 mg/Kg; <i>n</i> = 7), ZA (100 mg/Kg; <i>n</i> = 5) and tivantinib+ZA (<i>n</i> = 5). (<b>A</b>) Ventral images from a representative nude mouse for each group 24 days after implant. Pseudocolor scale bars are consistent for all images of ventral views, in order to show relative changes at metastatic sites over time. (<b>B</b>) Average growth of bone metastasis in the hindlimbs of controls and treated mice: photon counts from the hindlimb (right and left) regions of tumor-bearing mice were quantified and displayed over time. The data are presented as mean ± SE. **, p<0.005; ***, p<0.0001. (<b>C</b>) The presence of tumor-induced osteolytic lesions were detected by weekly micro-CT scans. Representative 3D reconstruction of micro-CT images of the hindlimbs of controls and treated mice at day 24 after xenografting are reported. Similar results were obtained in the second experiment conducted independently. Red circles indicate osteolysis, the circle’s size was proportional to the extent of bone lesion. Numbers at the bottom of the images represent the number of mice. (<b>D</b>) Kaplan-Meier survival plot of the survival rate of 1833/TGL xenografted controls and treated-mice.</p

Antitumor activity of tivantinib and ZA alone and in combination against subcutaneous breast cancer xenografts.

<p>A breast cancer xenograft model was established using 1833 cells (7.5×10⁶) implanted s.c. into the flanks of female athymic nude mice (6-week old). When tumor volume reached 70 mm³ animals were randomized into 4 groups: tivantinib (300 mg/Kg), ZA (100 mg/Kg), tivantinib+ZA-treated mice and controls treated with vehicle (PEG 400∶20% vit. E TPGS solution [60∶40]). Tivantinib and vehicle were administered p.o. in a volume of 10 mL/kg of body weight daily until the end of the experiment. ZA was administered i.p. every 2 days starting 13 days after implant till the end of the experiment. (<b>A</b>) Tumor growth inhibition. Tumor size was measured using Vernier caliper twice a week until the animals were sacrificed after 27 days of treatment. Tumor weight was calculated by the formula: Tumor weight (mg) = (length×width2)/2. (<b>B</b>) Relative body weight of mice bearing subcutaneous tumor xenografts and treated with vehicle or drugs. Body weights were measured twice weekly. Results are expressed as mean ± SD; <i>n</i> = 9–10.</p